Real-Time PCR

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Questions and Answers

What is Real-Time PCR?

A technique for visualizing DNA sequences without amplification

A process for amplifying target DNA using conventional PCR techniques

A method for determining the presence of target DNA after the PCR reaction

A modification of conventional PCR where product accumulation is quantified in real time (correct)

What is the main difference between Real-Time PCR and conventional PCR?

Real-Time PCR does not require a thermal cycler, unlike conventional PCR

Conventional PCR can quantify product accumulation 'in real time'

Conventional PCR is faster than Real-Time PCR

Real-Time PCR allows for quantification of product accumulation during the reaction, while conventional PCR does not (correct)

What can be determined using Real-Time or Quantitative (qPCR)?

The size of the DNA sample used in the PCR reaction

The temperature at which the PCR reaction occurs

The type of DNA polymerase used in the PCR reaction

The amount of target sequence or gene present in a sample (correct)

What is the mechanism behind SYBR® Green detection in qPCR?

It binds non-specifically to dsDNA and emits fluorescence upon intercalating with newly synthesized DNA Signup and view all the answers

What is the function of TaqMan® probe in qPCR?

It uses hydrolysis probes with specific sequences designed to bind downstream of the qPCR primers Signup and view all the answers

What happens to the TaqMan® probe during qPCR?

It is cleaved by DNA polymerase, enabling the reporter molecule to emit a fluorescent signal Signup and view all the answers

What is the Cycle Threshold (Ct) in qPCR?

The cycle number at which the fluorescent signal reaches the threshold line Signup and view all the answers

What does the Threshold line represent in qPCR?

The maximum level of fluorescence that can be considered as background Signup and view all the answers

What does the Baseline (negative control) represent in qPCR?

A successful negative control where no fluorescence is detected Signup and view all the answers

What does a low Ct value in qPCR indicate?

High abundance of the target sequence Signup and view all the answers

What is typically expected in samples with high Ct values in qPCR?

Low amounts of the target sequence Signup and view all the answers

In qPCR, what is the general relationship between Ct values and target sequence abundance?

Inverse relationship Signup and view all the answers

Quel est le rôle d'un gène de référence dans la qPCR?

Il s'agit d'un contrôle endogène présent à une concentration constante dans tous les échantillons et qui ne change pas en réponse à des conditions biologiques. Signup and view all the answers

Pourquoi est-il important que le gène de référence soit présent à une concentration constante dans tous les échantillons?

Pour permettre une comparaison précise de l'expression du gène cible entre les échantillons. Signup and view all the answers

Quel est le rôle de la sonde TaqMan® dans la qPCR?

Elle est utilisée pour détecter spécifiquement l'amplification du gène cible pendant la qPCR. Signup and view all the answers

What is required for each reaction in Absolute Quantitation analysis?

A standard of known concentration for the Reference Gene Signup and view all the answers

What is used to quantitate the concentration of unknown experimental samples in Absolute Quantitation analysis?

The standard curve generated using the log concentrations and the Ct value Signup and view all the answers

What is often identified using Absolute Quantitation analysis in qPCR?

DNA copy numbers Signup and view all the answers

What method is used to express the ratio between the Reference Gene and the Target Gene in qPCR?

Delta-delta Ct method Signup and view all the answers

Why is it crucial for the Reference Gene to remain unchanged in relative quantitation analysis?

To prevent erroneous results Signup and view all the answers

What does the accuracy of relative quantitation depend on in qPCR?

The Reference Gene (RG) Signup and view all the answers

What is one of the uses of qPCR in Pharmaceutical Biotechnology Quality Control in Biopharmaceutical Manufacturing?

Monitoring the presence of host cell DNA or RNA in biopharmaceutical products Signup and view all the answers

What does qPCR help to ensure in Viral Safety Testing of biopharmaceutical products?

Absence of viral contaminants, especially in products produced in cell lines Signup and view all the answers

In what way does qPCR contribute to Toxicology Studies of pharmaceuticals?

Assessing the effects of pharmaceuticals on gene expression in various tissues and cell types Signup and view all the answers

What is reverse transcription PCR (RT-PCR) used for?

Synthesizing cDNA from mRNA template Signup and view all the answers

What is the function of complementary DNA (cDNA) in RT-PCR?

It is synthesized from mRNA template Signup and view all the answers

Which enzyme is used to synthesize cDNA in RT-PCR?

Reverse transcriptase Signup and view all the answers

What is necessary to start the experiment with in order to clone the human gene for the insulin protein?

Human pancreatic islet cells actively expressing the insulin gene Signup and view all the answers

What is the specific starting material required for cloning the human gene for the insulin protein?

Human pancreatic islet cells Signup and view all the answers

Which type of cells should actively express the insulin gene for cloning the human gene for the insulin protein?

Pancreatic islet cells Signup and view all the answers

What is the purpose of Oligo d(T) Affinity chromatography column?

To enrich mRNAs present in the total RNA population without risking degradation of the RNA Signup and view all the answers

Why is RNA rapidly degraded in the cell?

Due to the presence of ribonucleases (RNAse) Signup and view all the answers

What type of RNA does the total RNA fraction obtained after cell lysis contain?

All 4 types of RNA Signup and view all the answers

What is an important consideration for the quality of RNA in quantification?

Absence of genomic DNA (gDNA) contamination and RNase activity Signup and view all the answers

How should the RNA sample be checked for quality?

Using a spectrophotometer Signup and view all the answers

Why is it important to ensure the RNA sample is free of genomic DNA (gDNA) contamination?

To prevent interference with RNA quantification Signup and view all the answers

What is the function of oligo (dT) primers in reverse transcription?

Bind to the poly-A tail of mRNA to amplify the entire mRNA into cDNA Signup and view all the answers

What is the role of random primers in reverse transcription?

Bind at different locations throughout the mRNA for cDNA synthesis Signup and view all the answers

How long is the typical incubation period for the enzymatic synthesis of cDNA from RNA template in reverse transcription?

30-60 minutes Signup and view all the answers

Study Notes

Real-Time PCR (qPCR)

Real-Time PCR (qPCR) is a laboratory technique used to amplify and quantify specific DNA sequences in real-time.
The main difference between Real-Time PCR and conventional PCR is the ability to quantify the amplified DNA in real-time.

Applications of Real-Time PCR

Real-Time PCR can be used to determine the presence or absence of a specific DNA sequence, quantify the amount of a specific DNA sequence, and detect genetic variations.
Real-Time PCR can also be used to analyze gene expression, detect pathogens, and identify genetic mutations.

Mechanism of SYBR Green Detection

SYBR Green is a fluorescent dye that binds to double-stranded DNA.
As the polymerase synthesizes new DNA strands, SYBR Green binds to the amplified DNA, and the fluorescence emitted is proportional to the amount of amplified DNA.

TaqMan Probe

A TaqMan probe is a fluorescently labeled oligonucleotide that is specific to the target DNA sequence.
The TaqMan probe is degraded by the 5' nuclease activity of the Taq polymerase during PCR, releasing the fluorescent dye.
The amount of fluorescence emitted is proportional to the amount of amplified DNA.

Cycle Threshold (Ct) and Baseline

The Cycle Threshold (Ct) is the cycle at which the fluorescence signal is detectable above the background signal.
The Threshold line represents the level of fluorescence that is considered significant.
The Baseline (negative control) represents the fluorescence signal in the absence of the target DNA sequence.

Ct Value and Target Sequence Abundance

A low Ct value indicates a high abundance of the target DNA sequence.
A high Ct value indicates a low abundance of the target DNA sequence.

Reference Gene

A reference gene is a gene that is present at a constant concentration in all samples.
The reference gene is used as an internal control to normalize the expression of the target gene.

Absolute Quantitation Analysis

Absolute Quantitation analysis requires a standard curve to relate the Ct value to the concentration of the target DNA sequence.
The concentration of unknown samples is determined by comparing their Ct values to the standard curve.

Relative Quantitation Analysis

Relative Quantitation analysis compares the expression of the target gene to that of a reference gene.
The ratio of the target gene to the reference gene is calculated using the 2^(-ΔΔCt) method.

Pharmaceutical Biotechnology Applications

qPCR is used in Pharmaceutical Biotechnology Quality Control to detect and quantify contaminants in biopharmaceutical products.
qPCR helps ensure the safety of biopharmaceutical products by detecting viral contaminants.
qPCR is used in Toxicology Studies to detect and quantify gene expression in response to pharmaceuticals.

Reverse Transcription PCR (RT-PCR)

Reverse Transcription PCR (RT-PCR) is used to amplify RNA sequences.
Complementary DNA (cDNA) is synthesized from RNA using reverse transcriptase.
cDNA is used as a template for PCR amplification.

Cloning the Human Insulin Gene

The human insulin gene can be cloned using mRNA isolated from cells that actively express the insulin gene.
Oligo d(T) Affinity chromatography is used to isolate mRNA from total RNA.
RNA is rapidly degraded in the cell, so it is essential to work quickly and carefully to preserve the RNA.

RNA Quality and Primers

RNA quality is critical for successful reverse transcription and PCR amplification.
RNA should be checked for quality using agarose gel electrophoresis or bioanalyzer.
Oligo d(T) primers and random primers are used in reverse transcription to synthesize cDNA.
It is essential to ensure the RNA sample is free of genomic DNA (gDNA) contamination.

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Description

Test your knowledge of Real-Time PCR, a modification of conventional PCR used to quantify and visualize product accumulation in real time as the reaction progresses.